Generation and phenotypic analysis of protein s-deficient mice Protein
S (PROS) is an important natural anticoagulant although its biological
functions are not precisely known. To investigate further the role of PROS in vivo, we generated PROS null mice through conditional gene targeting. Generated mice exhibited one floxed allele in which the exons 3-7 in the PROS genomic locus were flanked by two loxP sites. They were bred with a Cre-deleter strain (Nestin-Cre) to generate mice with one WT allele and one Cre-recombined (Delta) allele (PROS Delta/+ mice). In the Delta allele, Cre-mediated recombination resulted in a shift of the reading frame and generated a null allele. PROS Delta/+ mice were viable, with no apparent abnormalities, and reproduced normally. These mice had reduced PROS plasma levels and activated protein C-cofactor activity, as compared to WT mice. Moreover, PROS Delta/+ mice showed higher mortality than WT in a model of tissue factor-induced venous mboembolism
(P = 0.009, n = 8). At 20 min, 87.5% WT and only 12.5% PROSDelta/+ mice survived, respectively. No PROS/Delta/Delta neonate was obtained through mating of PROS/Delta/+ parents, suggesting that the homozygous deficiency in PROS is lethal. Genotyping of successive litters (153 mice) showed that 48 mice (31%) were WT and 105 (69%) were PROS/Delta/+.
Four E17.5 PROS/Delta Delta embryos were bruised in the head, and presented a prothrombotic and necrotic phenotype mainly in the brain and liver. Histological analysis demonstrated extensive bleedings in PROS/Delta/Delta brains, together with signs of tissue necrosis in the liver and loss of hepatocytes. PROS/Delta/Delta embryos likely presented disseminated intravascular coagulation as demonstrated by interstitial fibrin deposition and an increased number of megakaryocytes in the liver suggesting peripheral thrombocytopenia. Thus, heterozygous PROS deficiency in mice was associated with a prothrombotic phenotype further exacerbated and incompatible with life in mice with total deficiency in PROS.